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Nicotinamide adenine dinucleotide (NAD+) is a cofactor – a molecule that assists enzymes in chemical reactions – that plays an essential role in multiple physiological processes such as energy metabolism, DNA repair, and immune activation. It is essential for the activity of sirtuins, a class of enzymes involved in longevity, and poly ADP-ribose polymerase (PARP), a family of DNA-repair enzymes. Cellular NAD+ production declines with age, however, and its depletion has been implicated in the onset and progression of multiple age-related conditions such as metabolic dysregulation and neurodegenerative disease.

NAD+ can be synthesized in the body from a variety of dietary sources including tryptophan (an amino acid) and the three forms of niacin (vitamin B3) – nicotinamide (NAM, also called niacinamide), nicotinic acid (NA), and nicotinamide riboside (NR) – commonly referred to as niacin equivalents.[1] Of these three, NA is the primary source of NAD+. These precursors are not equally allocated throughout the body, exhibiting preferential distribution among the blood, brain, gut, and other organs.

Niacin is found in a variety of foods, including beans, milk, meat, and eggs. Nicotinamide mononucleotide (NMN), another dietary precursor of NAD+, can also be found in a variety of foods, such as broccoli, avocado, and beef.[2] Numerous animal and human studies have shown that increasing NAD+, either through dietary intake of a precursor or via direct supplementation, may be useful in preventing or reversing age-related disease.[3]

Biosynthesis & recycling

The body's predominant source of NAD+ is the NAD+ salvage pathway. This is, in part, because enzymes such as sirtuins and PARPs consume NAD+ molecules, depleting cellular levels and generating nicotinamide (a niacin equivalent) as a byproduct. The salvage pathway converts nicotinamide to nicotinamide mononucleotide (NMN) via the enzyme nicotinamide phosphoribosyltransferase (NAMPT). Through multiple steps, NMN is eventually converted to NAD+.[4] It is important to note that this enzyme is subject to feedback inhibition by NAD+ levels, which means that after a certain level of NAD+ is reached, the salvage pathway will stop producing NAD+.

Energy sensor

NAD+ is critical for energy generation within cells. In the mitochondria, NAD+ is a cofactor for metabolic enzymes involved in oxidative phosphorylation, a process in which energy is produced in the form of ATP via the transfer of electrons from electron carriers, such as NAD+, to oxygen. Outside the mitochondria, NAD+ is a cofactor for enzymes involved in glycolysis – the production of energy in the form of ATP from glucose.[5]

When cellular energy levels are low, such as during exercising, fasting, or caloric restriction, cellular NAD+ levels rise, and the ratio of NAD+ to its reduced form, NADH, increases, thereby serving as a "sensor" to turn on energy-generating pathways and activate enzymes such as sirtuins.[6] Conversely, the depletion of NAD+ due to DNA damage or aging may result in the suppression of NAD+-dependent ATP generation and possible cellular energy crisis.

NAD+ status in aging and potential for intervention. Cellular levels of NAD+ increase under conditions of energy stress, such as caloric restriction or exercise; via supplementation of NAD+ precursor molecules, NR and NMN, which "boost" synthesis; or via sirtuin-activating compounds (STACs), such as resveratrol or pterostilbene. NAD+ levels decrease during aging due to increased NAMPT activity, as well as age-related cellular demand for PARP-directed DNA repair, immune function, and inflammation.

Aging

NAD+ depletion has been associated with the hallmarks of aging, such as decreased autophagy, increased DNA damage, increased mitochondrial dysfunction, and dysregulated nutrient sensing.[3] Depletion of NAD+ may predispose organisms to the development of a variety of age-related diseases including neurodegenerative diseases, metabolic diseases, and cancer.[7] NAD+ levels have been shown to decrease with age in multiple species, including humans.[8] [9] [10]

In contrast, NAD+ levels increase under conditions that promote greater healthspan or lifespan such as exercise or calorie restriction.[6] [11] Furthermore, NAD+ restoration has been shown to increase lifespan in lower-level organisms such as yeast and worms, as well as in rodents.[12] [13] In a mouse model of Alzheimer's disease, increasing NAD+ through precursors NR or NMN ameliorated mitochondrial dysfunction and neuronal cell death, thereby slowing cognitive decline.[14] [15]

Taken together these findings suggest that NAD+ plays a critical role in aging. Specifically, the reduction of NAD+ levels commonly observed in aging is thought to be a combination of decreased synthesis, increased consumption, and increased degradation.[16] Depletion of NAD+ can result in unrepaired DNA damage since PARP-1 requires NAD+ to function. Furthermore, immune cells activated upon injury and inflammation have a high energy demand and may deplete NAD+ pools. Therefore, increased DNA damage and inflammation commonly observed in aging may decrease NAD+ and potentiate aging.[17]

Physiological responses of NAD+

NAD+ is involved in multiple physiological processes such as DNA repair, regulation of inflammation, autophagy, and energy metabolism, all of which play important roles in the regulation of healthspan and aging.

DNA damage, and DNA repair

As organisms age, accumulation of DNA damage may drive processes of aging and the development of age-related diseases. PARP enzymes, however, detect DNA damage and initiate a repair response by signaling other DNA-repair enzymes. In this way, PARP likely influences longevity. For example, PARP activity in the white blood cells of multiple mammalian species is a predictor of maximum lifespan.[18] Furthermore, findings from a study in which activity of PARP-1 was measured across multiple mammalian species demonstrated that the higher the PARP-1 activity, the longer the lifespan.[19] The difference in PARP-1 activity between the longest-lived mammals tested (humans) and the shortest-lived mammals tested (rats) was 5-fold. Remarkably, a study that used cell lines derived from centenarians (people older than 100 years of age) demonstrated that the cells' PARP activity was considerably higher than cell lines from much younger adults.[20] PARP-1 activity has also been correlated with maximum lifespan in mammals.

Skin is particularly vulnerable to the effects of aging. A study that examined human skin samples from 49 people between the ages of 15 and 77 years found that with age, PARP-1 activity increased, but NAD+ levels decreased.[21] Excessive DNA damage has also been shown to decrease NAD+ to 20 to 30 percent of its normal levels.[22] [23] As we age and accumulate more DNA damage, our cells increase their demand for DNA repair. The chronic reduction of NAD+ levels may eventually lead to the depletion of NAD+. The hyperactivity of PARP-1 and subsequent consumption of NAD+ may limit the activity of other NAD+-consuming enzymes such as sirtuins.[24]

Increases to the NAD+ pool promote sirtuin activity. SIRT1 activation leads to improved insulin function in the liver, improved cognitive function in the brain, and increased mitochondrial biogenesis in skeletal muscle. SIRT3 activation leads to improved insulin sensitivity and lowers inflammation in the liver. Loss of sirtuin activity during aging is associated with altered NAD+ status.[4]

Sirtuin activation

NAD+ is a substrate for the sirtuin proteins, a family of enzymes involved in a variety of metabolic processes. Sirtuins utilize NAD+ to remove specific chemical structures called acetyl groups – a process called deacetylation – from cellular proteins to control transcriptional regulation, energy metabolism, circadian rhythms, DNA repair, and cell survival. As described above, the ratio of NAD+ to its reduced form, NADH, increases under conditions of low cellular energy, switching on sirtuin expression and activity. Sirtuins must respond to dynamic changes in NAD+ to trigger appropriate adaptive responses. Broad loss of sirtuin activity, potentially due to a reduction in NAD+, is typically observed with aging, and many animal models demonstrate that decreased SIRT1 activity may promote the pathogenesis of both cardiovascular and neurological diseases.[25] [26]

Mitochondrial function

Mitochondria are classically thought of as the powerhouses of the cell because they generate energy in the form of ATP. They are also deeply involved in cellular metabolism and signaling pathways. Mitochondrial dysfunction is a hallmark of the aging process and may lead to reduced energy production, disruption of cellular metabolism, and progression of age-related diseases. Mitochondrial maintenance through mitophagy (selective elimination of defective mitochondria) and mitochondrial biogenesis (synthesis of new mitochondria) is key to maintaining cellular homeostasis and health.[27]

NAD+ has been shown to be an important regulator of mitochondrial maintenance. The rise of NAD+, due to fasting, for example, has been shown to induce mitochondrial biogenesis.[28] Furthermore, raising NAD+ levels in old mice can restore mitochondrial function to those of a young mouse.[29]

Circadian rhythm

The circadian system, which is composed of multiple cellular clocks found in all cells throughout the body, orchestrates the regulation of gene expression that coordinates metabolic programs needed to support bodily functions. NAD+ levels cycle in a 24-hour rhythm and are regulated by the core clock genes CLOCK and BMAL1.[30] These genes regulate the circadian expression of NAMPT, the rate-limiting enzyme in the NAD+ salvage pathway.[31] Furthermore, NAD+ itself can directly regulate circadian rhythm through the activation of sirtuins.[32] [33] Thus, NAD+ is extensively involved in the regulation of the circadian clock, which, when functioning properly, helps maintain homeostasis and health.

Energy metabolism

NAD+ plays a key role in energy metabolism by accepting and donating electrons through the back-and-forth processes of reduction and oxidation – often referred to as redox reactions. These alternating conversions of NAD's oxidized form (NAD+) to its reduced form (NADH) play significant roles in the reactions associated with the metabolism of glucose and fatty acids and the formation of ATP. Since both the oxidized and reduced forms of NAD+ are essential for these linked sets of reactions, cells maintain significant concentrations of both NAD+ and NADH. Without NAD+ and its related molecules, life would cease to exist.[34]

NAD+ boosters and supplementation

Boosting levels of NAD+ has been shown to improve lifespan and healthspan in premature aging animal models, prompting further investigation of NAD+ supplementation in humans to treat and prevent age-related diseases.[7] In particular, two key NAD+ precursors, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), have been extensively studied and shown to ameliorate age-associated diseases in animals.1 NR and NMN have been shown to be well tolerated at high doses and to effectively raise NAD+ levels in rodents. Currently, only NR has been shown to raise plasma NAD+ levels and may possibly have beneficial health effects in humans.[35] [36] Generally, cells do not directly take up NAD+, however, and studies in humans regarding the efficacy and safety of NAD+ supplementation by intravenous infusion are lacking.

NAD+ has poor bioavailability. Animal studies have shown that upon ingestion, NAD+ is broken down into the precursor nicotinamide primarily, but also NR and NMN, before being absorbed.[37] While oral bioavailability of NAD+ is low, it is possible that intravenous NAD+ infusion may be used to bypass the digestive system. Unfortunately, no mammalian NAD+ transporter has been identified, and with the exception of the brain and the heart, extracellular NAD+ has not been shown to be taken up into tissues. For example, NAD+ administered by intraperitoneal injection increased NAD+ in some brain regions in mice.[38] Similarly, mice injected with a high dose of NAD+ had increased levels in the heart and protection from cardiac hypertrophy.[39] In-vitro studies have also shown some cell types might be able to transport extracellular NAD+ across the plasma membrane.[40]

Nicotinamide riboside versus nicotinamide mononucleotide

Levels of NAD+ fluctuate throughout the body's tissues, based on cellular consumption and precursor or substrate availability. The highest variation occurs in the small intestine and spleen and the lowest variation occurs in the skeletal muscle.

One study used isotope tracers to track NAD+ flux in mice, relative to its source.[41] When oral tryptophan was the source, NAD+ was produced in the liver for local use, where it was broken down into nicotinamide, as described above. Nicotinamide was then exported to other tissues, such as the muscles, brain, and gut, which used salvage pathways to convert it back to NAD+.

The authors of the study investigated whether oral NR and NMN would be exported to other tissues where they would then form NAD+ directly, bypassing the NAMPT feedback inhibition. Using isotope tracers that allowed them to distinguish NAD+ made directly from NR or NMN versus NAD made from nicotinamide, NR, or NMN, they found that a low oral dose of the precursors (50 milligrams per kilogram body weight, mg/kg/bw) produced very low levels of NAD+ made directly from NR and NMN in the liver but not in other tissues. Low levels of nicotinamide-derived NAD+, on the other hand, were found in the kidneys, muscles, and brain, formed from the salvaged nicotinamide in NR and NMN.

A higher oral dose of 200 mg/kg/bw of NR showed no difference compared to a low dose in terms of making direct NAD+ in any tissues other than the liver. However, more of the nicotinamide-derived NAD+ was found in the kidney, muscle, and brain than at a lower dose of 50 mg/kg/bw.

When NR and NMN were given intravenously at varying doses (50 mg/kg/bw and 500 mg/kg/bw), directly-produced NAD+ was found in the liver, kidney, and muscle in a dose-dependent manner. However, the only NAD+ detected in the brain was that which was salvaged from nicotinamide, suggesting that neither NR nor NMN crosses the blood-brain barrier. It is noteworthy that a head-to-head comparison of identical doses of injected NR and NMN produced more NAD+ made directly from NR in the liver, kidney, and particularly in the muscle, compared to NMN.

Conclusion

NAD+ mediates multiple physiological processes such as energy metabolism, DNA repair, and immune activation. Without NAD+, life would cease to exist. As we age NAD+ levels fall and this is associated with the progression of age and age-related diseases. Boosting NAD+ has successfully been shown to improve both lifespan and healthspan in multiple animal models as well as improving age-related diseases in humans.

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